We previously reported that over-expression of Pitx2a in HeLa cells resulted in the accumulation of p53 and p21/cip1/walf1, leading to cell cycle arrest at G1/G0. However, it is not known how Pitx2a induced p53 and p21 protein accumulation in HeLa cells. In normal cells and tissues, the ubiquitination and proteasome-dependent degradation of p53 are mediated by ubiquitin protein ligases mdm2 and p300/CBP, maintaining the p53 protein at an appropriate level. In the presence of HPV E6 protein, E6AP (E6-associated protein), but not mdm2, is used as the ubiquitin protein ligase to target the tumor suppressor protein p53 for degradation. However, RT-PCR and northern blot analysis indicated that E6 and the E6AP mRNA levels in HeLa cells were not significantly affected following Ptx2a expression for 24h, suggesting that p53 accumulation induced by Pitx2a in HeLa cells was not due to the repression of HPV E6 and cellular E6AP expression. Furthermore, over-expression of E6AP protein in HeLa cells did not significantly affect the induction of p53 accumulation by Pitx2a. These results suggested that Pitx2a might directly block the ubiquitination and degradation of p53 by E6 and E6AP. Transfection of Pitx2a into cell lines containing high levels of functional mdm2, such as U2OS, did not result in the accumulation of p53, suggesting that Pitx2a did not affect mdm2-mediated p53 degradation. Co-immunoprecipitation experiments indicated that Myc-tagged p53 and GFP-tagged Pitx2a could interact with each other in HeLa cells. This interaction does not require the presence of the HPV E6 oncoprotein, as Pitx2a can still bind to p53 when they were cotransfected into HPV-negative Sao-2 cells. Deletion of either the homeodomain or 40 amino acids in the carboxyl-terminal region of Pitx2a abrogates the ability of Pitx2a to induce p53 accumulation in HeLa cells. In addition, over-expression of Pitx2a caused the ubiquitination and degradation of transfected E6AP. Our data suggest that Pitx2a increases p53 stability by forming a complex with p53 and accelerating the ubiquitination and degradation of E6AP, leading to the accumulation of p53. Previous reports demonstrated that ubiquitous ablation of NMHC II-B in mice (B-/B-) resulted in defects in both the brain and heart, leading to lethality between E14.5 and E18.5. The presence of both severe cardiac and brain defects in B-/B- mice raised the possibility that at least some of the abnormalities found in these organs could be secondary to the malfunction of the other organ, rather than directly due to the absence of NMHC II-B. We, therefore, utilized loxP/Cre strategy to specifically delete exon II of NMHC II-B in a tissue-specific manner. A mouse line, B:loxP/loxP, in which exon II of NMHC II-B was flanked by loxP sites, was generated. NesCre, a transgenic mouse line harboring the Cre recombinase expression cassette under control of the nestin promoter, was crossed to B:loxP/loxP mice to obtain B:loxP/loxP NesCre, which allowed removal of NMHC II-B in the brain, but not the heart. The B:loxP/loxP NesCre mice are smaller than wild-type littermates, develop severe hydrocephalus, and demonstrate abnormal neuronal cell migration without showing any cardiac defects. B:loxP/loxP mice were also crossed to a transgenic mouse line, alpha-MHC-Cre (MHCCre), in which the Cre expression cassette is driven by the alpha-cardiac myosin heavy chain promoter, to generate B:loxP/loxP MHCCre mice, in which NMHC II-B was specifically ablated in the heart. B:loxP/loxP MHCCre mice are presently being characterized, but have demonstrated cardiac ventricular septal defects and no brain abnormalities to date. Thus, the defects in B-/B- mice appear to be directly caused by the loss of NMHC II-B in each organ and are independent of the defects generated in the other organ.